MA5-13070

antibody from Invitrogen Antibodies

Targeting: EGFR ERBB, ERBB1

Provider product page for MA5-13070

Western blot

Immunoprecipitation

Antibody data

Antibody Data
Antigen structure
References [70]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [5]
Immunohistochemistry [2]
Flow cytometry [2]
Other assay [9]

Submit

Product number: MA5-13070 - Provider product page
Provider: Invitrogen Antibodies
Product name: EGFR Monoclonal Antibody (H11)
Antibody type: Monoclonal
Antigen: Other
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: H11
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

EGFR protein structure - MA5-13070 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Epidermal Growth Factor Receptor (green) showing staining in the membrane of A431 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an Epidermal Growth Factor Receptor monoclonal antibody (Product # MA5-13070) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Epidermal Growth Factor Receptor (green) showing staining in the membrane of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with an Epidermal Growth Factor Receptor monoclonal antibody (Product # MA5-13070) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of EGFR was performed using 90% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with EGFR Mouse monoclonal antibody (Product # MA5-13070) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of EGFR was achieved by transfecting A-431 cells with EGFR specific siRNA (Silencer® select Product # s563, s564 and s565). Immunofluorescence analysis was performed on A431 cells (untransfected, panel a,d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with EGFR specific siRNA (panel c,f) Cells were fixed, permeabilized, and labelled with EGFR Mouse monoclonal Antibody (Product # MA5-13070, 5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal was observed upon siRNA mediated knockdown (panel c,f) confirming specificity of the antibody to EGFR (green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of EGFR was performed using 70% confluent log phase A-431 cells (WIld type, panels a,d), CAS9 control (panels b,e) and EGFR Knockout (panels c,f). The cells were fixed, permeabilized, and labelled with EGFR Mouse Monoclonal Antibody(Product # MA5-13070, 5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000). Nuclei (blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal was observed in EGFR Knockout cells (panel c,f) confirming specificity of the antibody to EGFR(green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Epidermal Growth Factor Receptor using Epidermal Growth Factor Receptor Monoclonal Antibody (Product # MA5-13070) on Native Human A431 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1 EGF promotes the EGFR--sortilin interaction. a A549 cells grown in complete cell culture media were stimulated or not with EGF (50 ng/mL) for 30 min. Immunoprecipitations (IP) were performed using anti-EGFR antibody, and the immunocomplexes were immunoblotted (IB) using anti-sortilin antibody (top). In parallel, immunoblots for P-EGFR, EGFR, sortilin, P-ERK, and ERK were performed on whole-cell lysates (WCL); the isotypic lane Immunoglobulin G (IgG) represents the IP control. b Proximity ligation assays (PLA) were performed on A549 cells, non-stimulated or stimulated with EGF (50 ng/mL) for 2, 5, 15, and 30 min. Red spots indicate sites of proximity ligation assay amplification, reflecting the EGFR--sortilin interaction (white arrows). Scale bar, 10 um. c Quantification of PLA time course, in comparison with non-stimulated cells. d A549 cells were stimulated or not with EGF (50 ng/mL) for 30 min, and then co-immunolabeled for EGFR and markers of the early endosome (Rab5), the late endosome/lysosome (LAMP2) and the trans-Golgi network (TGN46). For sortilin labeling, A549 cells were transiently transfected with sortilin-GFP to adapt a set of functional antibodies, and then immunolabeled for the same markers. Scale bar, 5 um. e Quantitative analysis of EGFR or sortilin-GFP co-localization with the aforementioned organelle-specific markers. f A549 cells were transiently transfected with sortilin-GFP, and then stimulated with EGF (50 ng/mL) for 30 min. Next, cells were fixe

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 EGFR interacts with sortilin at the cell surface. a A549 cells were pretreated or not with the cell-permeable dynamin inhibitor Dynasore (40 uM) for 2 h, and then stimulated or not with EGF (50 ng/mL) for 15 min. Cells were immunolabeled for EGFR and the early endosome marker EEA1, and then analyzed by confocal microscopy. Scale bar, 10 um. Inset 4-3: Bright-field image of A549 cells; white arrows show EGFR clusters at the cell surface. Scale bar, 10 um. b A549 cells were pretreated or not with Dynasore (40 uM) for 2 h, and then stimulated or not with EGF (50 ng/mL) for 15 min. The cell lysates were analyzed by western blotting for P-EGFR and EGFR. c A549 cells were transfected with EEA1-GFP, pretreated with Dynasore (40 uM) for 2 h, then stimulated with EGF (50 ng/mL) for 30 min. Next, cells were co-immunolabeled for EGFR and sortilin. Scale bar, 10 um. The co-localization profile is shown in d . e Proximity ligation assays (PLA) were performed on A549 cells under the same conditions described above in a . Scale bar, 10 um. f PLA quantification in comparison with A549 non-stimulated cells. All values represent means +- SD, Student's t -test *** P < 0.001. Each experiment has been repeated at least three times

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 C-terminally truncated sortilin strongly interacts with EGFR at the cell surface independently of ligand stimulation. a HEK293 cells were transiently transfected with either full-length (FL) or C-terminally truncated (Deltac) sortilin-GFP. Cells were fixed and immunolabeled for the trans-Golgi network marker TGN46, and then analyzed by confocal microscope. Scale bar, 10 um. b Bars show the Mander's coefficient, indicating that FL-sortilin-GFP co-localized with TGN46 to a greater degree than Deltac-sortilin-GFP. c Bars show quantification of cell surface GFP intensity. d Strong interaction between EGFR and Deltac-sortilin at the plasma membrane. HEK293 cells were transiently co-transfected with EGFR and FL- or Deltac sortilin-GFP, and then fixed and immunolabeled for EGFR; immunofluorescence was analyzed by confocal microscopy. Scale bar, 10 um. e HEK293 cells were transiently co-transfected with EGFR and FL or Deltac sortilin-v5. Immunoprecipitations (IP) were performed using anti-v5 antibody, and immunocomplexes were analyzed by western blotting using anti-EGFR antibody. In parallel, immunoblots of EGFR and sortilin-v5 were performed on whole-cell lysate (WCL). f HEK293 cells were transiently co-transfected with EGFR-GFP and FL- or Deltac sortilin. Next, cells were stimulated or not with EGF (50 ng/mL) for 30 min and immunoprecipitated using anti-GFP antibody. Immunocomplexes were analyzed by western blotting for sortilin. In parallel, immunoblots of P-EGFR, EGFR

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 Loss of sortilin greatly perturbs EGFR internalization and dramatically promotes EGFR signaling. a A549 cells expressing shRNA targeting sortilin and control cells (pLKO) were stimulated with EGF (50 ng/mL) for 30 min, and then fixed and immunolabeled for EGFR. Immunofluorescence was analyzed by confocal microscopy. The ratio between whole-cell and intracytoplasmic EGFR intensities, reflecting EGFR membrane retention, is quantified in b . c Sortilin-depleted and control A549 cells (pLKO) were stimulated with EGF (50 ng/mL) over a 60 min time course. At each time point, cell surface EGFR was stained at 4 degC using Alexa Fluor 488 anti-EGFR, and then analyzed by flow cytometry. Curves represent mean fluorescence intensity in sortilin-depleted cells relative to the control. d Cell lysates from sortilin-depleted (sortilin shRNA) or control A549 cells (pLKO) were immunoblotted with the indicated antibodies. e A549 sortilin-depleted and control cells (pLKO) were stimulated with EGF (50 ng/mL) over a 60 min time course. Cell lysates were analyzed by western blotting for components of the canonical EGFR signaling pathway using the indicated antibodies. f Sortilin-depleted cells were transfected with control siRNA (Si co) or EGFR siRNA. Cell lysates were analyzed by western blotting with the indicated antibodies. g Representative histograms of cell proliferation, as determined by EdU incorporation. Sortilin-depleted cells transfected or not with EGFR siRNA and control A549 cel

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. EGFR expression is upregulated in PCa tissues. (A) Representative images of EGFR staining in PCa and adjacent prostate tissues. Scale bars, 100, 50 and 20 um. (B) The ratio of high EGFR expression and low EGFR expression between PCa and adjacent prostate tissues. (C) EGFR mRNA expression in PCa and adjacent prostate tissues was assessed via reverse transcription-quantitative PCR. (D) Western blotting results of EGFR and p-EGFR protein expression in PCa and adjacent prostate tissues. (E) The gray degree values of p-EGFR/EGFR western blotting results. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 EGFR family extracellular domain expression and purification in 293-F cells. (A) Time-course western blot (WB, anti-HER2 or anti-His-tag) of conditioned media (CM) indicating that the HER2 protein is expressed and secreted at high levels by day 5 post-transfection. The secreted IgG control vector is detected by the secondary HRP antibody. (B) SDS-PAGE and Coomassie staining of purified HER2 product following Ni 2+ -NTA purification. (C) WB of fractions from Ni 2+ -NTA purification. SDS-PAGE, Coomassie staining, and WB analysis of (D) EGFR and (E) HER3 following Ni 2+ -NTA purification. Lane descriptions are below each panel. FT is flow-through.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 Anchoring DNA nanotubes to EGFR receptors using AMDA. a Confocal micrographs of seeded nanotubes anchored to HeLa cells after EGFR AMDA and EGFR AMDA with BDC tag addition omitted (No BDC tag) (Supplementary Note S 26 ). Seeds labeled with atto647 (red) and nanotubes with Cy3 (green), streptavidin with Alexa488 (blue). Scale bar: 20 mum. b Three-dimensional reconstruction of a HeLa cell with seeded nanotubes attached to EGFR. Scale bar: 20 mum. c The average fluorescence intensities of HeLa cells with seeded nanotubes or seeds attached via AMDA and AMDA with BDC tag addition omitted (Supplementary Note S 27 , N = 10 random locations for each case were analyzed). d Three-dimensional projection images of HEK293 cells with attached seeded nanotubes after AMDA and AMDA with BDC tag addition omitted (Supplementary Note S 28 ). Scale bar: 20 mum. e Three-dimensional reconstruction of a HEK293 cell with attached seeded nanotubes. Scale bar: 5 mum. f Average numbers of seeded nanotubes on HEK293 cells after AMDA and after AMDA with BDC tag addition omitted (Supplementary Note S 29 , N = 10 random locations for each case were analyzed). The average numbers of attached seeds are shown in Fig. 2f . g Confocal micrographs of HeLa cells at different times after seed attachment using EGFR AMDA (upper panel) and maximum projection images of HeLa cells at different times after seeded nanotube attachment using EGFR AMDA (lower panel) (Supplementary Notes S 31 , S 32 ). Scale bar: 20 mu

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 CK5/6 and EGFR expression by immunohistochemistry. (A,B) CK5/6 positive/negative staining; (C,D) EGFR positive/negative staining. EGFR, epidermal growth factor receptor.